Previous studies showed that chromium nanoparticles (Cr-NPs) might be used as dietary compounds against some obesity-related disorders; however, there is little information on how these compounds influence the gut microenvironment. The aim of this study was to investigate whether the negative effects of a high-fat diet in the large intestine of rats might be mitigated by switching to a low-fat diet and supplementation with Cr-NPs. Microbiota sequencing analysis revealed that the main action of the Cr-NPs was focused on changing the gut microbiota’s activity. Supplementation with nanoparticles decreased the activity of β-glucuronidase and enzymes responsible for the hydrolysis of dietary oligosaccharides and, thus, lowered the concentration of short-chain fatty acids in the cecum. In this group, there was also an elevated level of cecal lithocholic acid. The most favorable effect on the regulation of obesity-related disorders was observed when a high-fat diet was switched to a low-fat diet. This dietary change enhanced the production of short-chain fatty acids, reduced the level of secondary bile acids, and increased the microbial taxonomic richness, microbial differences, and microbial enzymatic activity in the cecum. To conclude, supplementation of a high-fat diet with Cr-NPs primarily had an effect on intestinal microbial activity, but switching to a low-fat diet had a powerful, all-encompassing effect on the gut that improved both microbial activity and composition. Obesity3 . Our und3 , and a h3 . In our 3 , a high-3 . Some pr3 . Those p3  confirmeG. frondosa polysaccharide-chromium (III) exhibited excellent hypoglycemic and hypolipidemic activities in diabetic mice induced by high-fat diet feeding and streptozotocin (STZ) injection by regulating liver-related gene transcription and modulating the intestinal microbiota. Previous studies on Wistar rats even showed that the gut bacteria provided the first line of defense to the body by converting toxic Cr(VI) to less toxic Cr(III), and the latter may act similarly to a prebiotic [Pseudomonas sp. and least with E. coli. Antibiotic resistance also developed with Lactobacillus sp. [Bacillus subtilis to mice, manifested as enhanced regulation of body growth, increased tissue organic Cr concentrations, beneficially altered cecal microbiota, and enhanced insulin receptor expression to produce significant changes in plasma biochemistry. The recent work of Dworzański and coauthors [The effects of exposure to some metals on the gut microbiota remain poorly characterized. As this microbiota creates a critical interface between the external environment and the host’s cells, it may play an important role in host outcomes during exposure. Richardson et al.  observedrebiotic . Those allus sp. . Yang etllus sp.  reportedoauthors  noted, iThe development of nanotechnology has ushered in new trends in many fields. The term ‘nanoparticle’ refers to a material with a particle size of less than 100 nm. It is believed that the lack of electric charge and the small size of chromium nanoparticles allows their properties to be modified in living organisms and their bioavailability to be improved relative to their standard counterparts. Recently, nanoparticles have emerged as important players in modern science, including the nutritional status of the host in health and disease. In the last decade, numerous nutrition experiments have also been carried out using metal nanoparticles, showing that the biological response of the organism depended on the size of the particles, the method by which they were produced, the dosage applied, and the length of administration . Li et aIn the present study, the main intention was to provide new insight with regard to the question of whether negative large intestinal effects associated with chronic consumption of a high-fat diet could be mitigated by switching to a low-fat diet accompanied by dietary chromium addition. Taking into account an urgent need to establish the true action of dietary nanoparticles in the gastrointestinal tract, paramount attention was given to chromium nanoparticles, and their action was compared to that of the commonly used dietary supplement chromium picolinate. The present study assessed the negative or positive impact of dietary habit change as well as supplemental Cr on the microbiota living in the lower gut of obese hosts.2/g (mean surface area), and 8.9 g/cm3 (true density). The purity of the chromium picolinate  was 98%.Chromium nanoparticles  were characterized as follows: 99.9% nanoparticles, 70 nm (mean size), 7 mMale outbred Wistar rats (Cmdb:Wi CMDB) were used in the present study (n = 84). The in vivo experimental schema was approved by the National Ethics Committee for Animal Experiments . Rats were kept individually in cages with constant comfortable temperature 22 ± 1 °C), relative humidity 60 ± 5%, a 12 h day/night period, and a ventilation rate of 15 air changes per hour. The study lasted 18 weeks and consisted of two 9-week periods, namely an initial and experimental period. Two types of diet were applied to rats: a low-fat diet  and a high-fat diet . Moreover, two forms of dietary additional chromium were used: standard Cr-picolinate and novel Cr nanoparticles  at the pharmacologically relevant dose of 0.3 mg/kg body weight. The details of the experimental dietary protocol were described in recently published work aimed at hepatic metabolism  ± 1 °C, . During After the 18-week feeding, the animals were deprived of feed for 12 h and then anesthetized with an i.p. mixture of ketamine and xylazine  in physiological saline. The rats were euthanized by cervical dislocation. After laparotomy, the cecum was dissected and weighed as the tissue and digesta masses.g for 10 min. The supernatant was loaded onto a capillary column  using an on-column injector. The initial oven temperature was 85 °C, which was raised to 180 °C at 8 °C/min and held for 3 min. The temperatures of the flame ionization detector and the injector were 180 °C and 85 °C, respectively. The sample volume for GC analysis was 1 μL. The concentrations of cecal putrefactive SCFAs (PSCFAs) were calculated as the sum of iso-butyric acid, iso-valeric acid and valeric acid. All SCFA analyses were performed in duplicate. Pure acetic, propionic, butyric, iso-butyric, iso-valeric, and valeric acids were obtained from Sigma Co. , and their mixture was used to create a standard plot and then to calculate the number of individual acids. This additional set of pure acids was included in each GC run of samples at five-sample intervals to maintain calibration.Samples of cecal contents were used for immediate analysis ), while the rest of the cecal digesta was transferred to tubes, frozen in liquid nitrogen, and then stored at −70 °C. In fresh cecal digesta, ammonia was extracted, trapped in a solution of boric acid in Conway’s dishes, and determined by direct titration with sulfuric acid. The dry matter of the digesta was determined at 105 °C. The concentrations of SCFAs in cecal digesta samples were determined by gas chromatography . The samples (0.2 g) were mixed with 0.2 mL of formic acid, diluted with deionized water, and centrifuged at 7211× v/v) formic acid in water (solvent A), and a mixture of methanol and acetonitrile at a proportion of 1 to 9 (v/v) (solvent B). The column temperature was set at 45 °C, the flow rate was 0.35 mL/min, and the gradient program was as follows: 0–8 min, 40% (v/v) B; 8–14 min, 50% (v/v) B; 14–15.3 min, 50–100% (v/v) B; 15.3–17.4 min, 40% (v/v). The injection volume was 1 µL. Mass data acquisitions were performed using LabSolutions 5.109 software (Shimadzu). Quantitative analysis was carried out with external standards (0.01–1 μM) that had been used to construct linear calibration curves with correlation coefficients of 0.994–0.997. All analyses were performed in triplicate for each sample.The bile acids (BAs) in the cecal digesta were assessed using a Shimadzu LC system  coupled to a Shimadzu LCMS-2020 mass spectrometer . Chromatographic separation was conducted using an Avantor ACE, C18-amide, 75 × 2.1 mm, 2.7-µm column by gradient elution with 0.01% (v/v) dilution of the cecal sample in 100 mM phosphate buffer (pH 7.0) after centrifugation at 7211× g for 15 min. Incubation was carried out at 37 °C, and p-nitrophenol was quantified at 400 nm and at 420 nm (o-nitrophenol concentration) after the addition of 2.5 mL of 0.25 M cold sodium carbonate. The enzymatic activity was expressed as μmol of product formed per hour per gram of digesta. The abovementioned procedure concerns the activities of bacterial enzymes released from bacterial cells into the cecal environment. The total enzymatic activity of selected enzymes, which includes the intracellular and extracellular enzyme activities, was also assessed. Therefore, another cecal digesta sample was mechanically disrupted by vortexing with glass beads  using the FastPrep®-24 homogenizer . The resulting mixture was centrifuged at 7211× g for 15 min at 4 °C. The supernatant was used for the enzyme assay described above. Intracellular enzyme activity was calculated by comparing total enzyme activity with the activities of bacterial enzymes secreted into the intestinal environment. To prepare the calculation formulas, the model curves for PNP and ONP  were used, and appropriate equations were obtained. All analyses were performed in duplicate.Bacterial enzyme activity was measured by the rate of p- or o-nitrophenol release from their nitrophenyl glucosides in the cecal digesta . The following substrates were used: p-nitrophenyl-α-D-glucopyranoside (for α-glucosidase), p-nitrophenyl-β-D-glucopyranoside (for β-glucosidase), α-nitrophenyl-α-D-galactopyranoside , o-nitrophenyl-β-D-galactopyranoside , p-nitrophenyl-β-D-glucuronide (for β-glucuronidase), and p-nitrophenyl-β-D-xylopyranoside (for β-xylosidase). The reaction mixture contained 0.3 mL of a substrate solution (5 mM) and 0.2 mL of a 1:10 .Two-way ANOVA and Student’s t test were used to analyze the results. The t test was applied to compare a single experimental group  with the control C group. Two-way ANOVA was applied to assess the effects of the main factors and the interaction between them . When ANOVA indicated significant treatment effects, means were evaluated using Duncan’s multiple range test. Data were checked for normality before statistical analysis was performed. Differences with Regarding the results of cecal bacterial sequencing, statistical analyses were performed according to a protocol from a previous study . Brieflyp < 0.05 vs. all experimental groups; t-test). Two-way ANOVA showed that rats fed the F diet had a higher final BW than that in the M treatment. The lowest fat tissue percentage  was noted in the MW and MP groups . The Cr × D interaction with regard to the lean tissue percentage showed that the FN group did not differ significantly vs. the MN and MP groups. The highest value for lean tissue percentage was attributed to the MW rats . The abovementioned data were not provided in the present work in the tables due to their presentation in the recent study by Fotschki et al. [The control C rats had significantly lower starting BW as they were fed the control low-fat diet . The cecal ammonia concentration was higher in all groups that in the C rats, except groups MW and MN. In comparison to the control C rats, the FW animals had a higher cecal pH value. The two-way ANOVA revealed that irrespective of additional Cr, the high-fat dietary treatment caused a significant decrease in the cecal concentrations of acetic, propionic, and butyric acid and total SCFAs compared to those in the M treatment . Regardless of the diet type, the addition of Cr-NPs caused a significant decrease in cecal acetic, propionic, butyric, iso-valeric, and total SCFA concentrations (p < 0.05). The t test analysis showed that the C group was higher than all the experimental groups with respect to the acetic and butyric acid and total SCFA concentrations in the cecal digesta . The acetic acid profile was reduced in the MP and FP groups vs. the C group. The FW, FP, and MN groups were characterized by enhanced propionic acid profile percentages (p < 0.05 vs. C). The butyric acid profile was diminished in the FW, FP, MN, and FN groups compared to the control C animals (p < 0.05).The two-way ANOVA showed that, regardless of Cr addition, the relative cecal tissue and digesta weights were diminished and the ammonia concentration and digesta pH values were enhanced in rats fed the high-fat diet in comparison to the M treatment  . The C rreatment . The prop < 0.05 vs. M treatment; p < 0.05 and p = 0.052, respectively, vs. M treatment). The dietary addition of Cr-NPs caused a tendency toward diminished cecal HDCA, regardless of the diet type (p = 0.091). A significant Cr × D interaction for the cecal lithocholic acid (LCA) level showed that the FW group was significantly higher than the MW group. The t-test revealed that only the FW and FN groups had higher ω-MCA cecal concentrations vs. the C group. The FW group had a decreased UDCA content, the FN group had diminished HDCA levels, and the FP group had an enhanced DCA cecal concentration in comparison to those of the C group (p < 0.05).Irrespective of addition of Cr, the high-fat dietary treatment (treatment F) substantially increased the cecal concentrations of β-muricholic acid (β-MCA), ω-muricholic acid (ω-MCA), cholic acid (CA), deoxycholic acid (DCA), and total analyzed bile acids . When Cr addition regardless of diet type was considered, Cr-NP addition resulted in decreased extracellular and total activities and release rate percentages of bacterial cecal α-glucosidase. Compared to the C group (t-test), the FW group had higher activity and the MN group had lower extracellular activity of α-glucosidase. The extracellular and total activities as well as the release rate of bacterial β-glucosidase were decreased in the groups fed the high-fat diet, irrespective of the addition of Cr. Two-way ANOVA showed that regardless of the diet type, dietary chromium nanoparticles caused a significant decrease in the extracellular activity and release rate of bacterial β-glucosidase in the cecal digesta (p < 0.05 vs. treatments without Cr and with Cr-Pic). The C group exceeded the FW, FP, MN, and FN groups with respect to the extracellular and total activities of bacterial β-glucosidase . A decrease in the extracellular and total activity of bacterial cecal α-galactosidase was evident with the F treatment, irrespective of Cr supplementation , except the MW and MP groups. The α-galactosidase release rate was decreased in groups MW, MN, and FN vs. the C group . Two-way ANOVA showed that regardless of Cr supplementation, an increase in the extracellular and total activities of bacterial cecal β-galactosidase was associated with the F treatment compared to the M (p < 0.05). Irrespective of diet type, Cr-NP supplementation caused a decrease in β-galactosidase extracellular and total activities as well as the release rate percentage . The t test revealed that the FW and FP groups were higher than the C group with respect to the extracellular and total activities of β-galactosidase. The release rate percentage was enhanced only in the FW animals .The extracellular activity of bacterial α-glucosidase in the cecal digesta was enhanced, while the intracellular activity was decreased with the F diet, irrespective of Cr addition (t-test), the FW and FP rats had higher extracellular activity, and the FW, FP, and MN animals had higher total activity of bacterial cecal β-glucuronidase (p < 0.05). A Cr × D interaction showed that the MW group was higher than the remaining groups with respect to β-xylosidase extracellular activity in the cecal digesta (p < 0.05). Regardless of the addition of Cr, the rats fed the high-fat diet had lower total activity of bacterial β-xylosidase (p < 0.05 vs. rats on the low-fat diet treatment). The t test revealed that all experimental groups, except MW, had lower extracellular activity in terms of bacterial cecal β-xylosidase than the C group.Dietary treatment with Cr-NPs caused a significant decrease in the extracellular activity and release rate of bacterial cecal β-glucuronidase compared to the treatment with no additional Cr and with Cr-Pic, irrespective of diet type . Comparep < 0.05 vs. C rats). Additionally, comparison between dietary treatment F (high-fat diet) and M (switching away from the high-fat diet) showed that the high-fat diet considerably decreased microbial taxonomic richness , the abundance changes in Eubacterium ventriosum group and Christensenellaceae were significantly elevated. Moreover, the beta diversity analyses at the genus level demonstrated that except for Adlercreutzia, all considered differences were related to bacterial genera from the Firmicutes phylum. However, the analyses at the phylum level in the group with the F dietary treatment showed considerably higher values of abundance changes only for Actinobacteriota, Bacteroidota, and Proteobacteria .Next-generation sequencing (NGS) analyses showed that supplementation with the examined types of Cr had no effect on the cecal microbiota profile . The maiProteobacteria and Bacteroidota at the phylum level as well as at the genus level of bacteria belonging to Firmicutes were considerably elevated in the groups with the F dietary treatment (18-week high-fat diet). These results are in agreement with other studies reporting that taxa from Bacteroides and Proteobacteria exhibited reduced growth in lower pH environments and that taxa from Firmicutes were more tolerant [The role of the gut microbiota is crucial in many metabolic diseases, including those linked to obesity . These mtolerant . Furthertolerant .Butyrivibrio abundance was considerably lower in comparison to that in the control diet. This gut bacterium is one of the main butyrate-producing strains [Another dietary factor that may modulate the activity and profile of the gut microbiota is supplementation with Cr ,18. In t strains . Changes strains  and inte strains .Other factors that might have a strong impact on intestinal microbiota activity are bile acids. The main role of these acids is to act as emulsifiers to facilitate the absorption of lipids from the diet . There aLactobacillus casei, Clostridium butyricum, and Bacillus mesentericus [The effect of switching from a high-fat diet to a standard diet on bacterial enzymatic activity in the cecum was mostly related to variation in the composition of bile acids, which are the main factors responsible for the changes in the intestinal microbial environment . In the ntericus , which antericus . TherefoSwitching to a low-fat diet accompanied by dietary Cr-NP addition may mitigate disorders induced by a high-fat diet in the large intestine environment. The analyses of the gut microbiota composition showed that the effect of the nanoparticles was limited to affecting the enzymatic activity of the microbiota. The favorable effect of nanoparticles was associated with a decrease in the extracellular activity of β-glucuronidase. Nevertheless, the most effective action in the regulation of gut-related disorders induced by a high-fat diet was shown by switching to a low-fat diet. This change in diet favorably increased the production of SCFAs, reduced the level of undesired secondary bile acids (DCA and LCA), favorably increased microbial taxonomic richness and differences, and, thus, increased microbial enzymatic activity responsible for the hydrolysis of oligosaccharides. To summarize, supplementation with chromium nanoparticles mainly affected intestinal microbial activity, whereas changing eating habits to a low-fat diet showed strong wide-ranging intestinal action, including an improvement in microbial activity and composition. Nevertheless, our previous study showed that chromium nanoparticles together with a switch away from high-fat/low-fiber dietary habits enhances the pro-healthy regulation of liver lipid metabolism and inflammation . Therefo


Macaca mulatta (rhesus monkey) genome, which is phylogenetically close species to humans, based on the genome coordinates of miRNAs and TEs. The expression of 5 out of 17 MDTEs that were exclusively registered in M. mulatta from the miRBase database (v22) was examined via quantitative polymerase chain reaction (qPCR). Moreover, Gene Ontology analysis was performed to examine the functional implications of the putative target genes of the five MDTEs.Transposable elements (TEs) are mobile DNA entities that can move within the host genome. Over long periods of evolutionary time, TEs are typically silenced via the accumulation of mutations in the genome, ultimately resulting in their immobilization. However, they still play an important role in the host genome by acting as regulatory elements. They influence host transcription in various ways, one of which as the origin of the generation of microRNAs (miRNAs), which are so-called miRNAs derived from TEs (MDTEs). miRNAs are small non-coding RNAs that are involved in many biological processes by regulating gene expression at the post-transcriptional level. Here, we identified MDTEs in the  TypicTEs are mobile DNA entities composed of repetitive sequences that induce genetic diversity via insertion into the host genome ,10. TEs M. mulatta, one of the most extensively studied non-human primates (NHPs). M. mulatta, also known as the rhesus monkey or rhesus macaque, belongs to the subfamily Cercopithecinae of Old World monkeys and is native to South Asian countries [M. mulatta is considered a natural intermediate model that bridges the evolutionary and genetic gap between humans and the experimental rodents often used in human clinical trials [M. mulatta made significant contributions during the COVID-19 pandemic as the most appropriate animal model for vaccine development [M. mulatta and humans. Understanding and studying these differences could aid in drug development and safety testing. Genome-based analysis revealed that the differences in the genomes of these two species are found in non-coding regions, such as UTRs, and are not in the highly conserved protein-coding regions [M. mulatta and humans can help researchers understand how they contribute to species-specific traits and adaptations. It may also provide an opportunity to overcome the challenges that interspecies differences can pose to drug testing for human clinical trials. However, a few studies have been conducted on miRNAs in M. mulatta, but no study on MDTEs.In our previous study, we investigated MDTEs and their implication to human diseases. Herein, we extend our study on MDTEs to ountries ,22. Theyountries ,24. For l trials ,26,27. Tl trials . In partelopment . However regions . Consequ regions . TherefoM. mulatta by intersecting the genomic coordinates of TEs and miRNAs. In addition, we compared identified M. mulatta MDTEs to human MDTEs based on our previous study [M. mulatta and humans and 17 MDTEs that were exclusively registered in M. mulatta based on the miRBase database (v22). We performed quantitative polymerase chain reaction (qPCR) to confirm the expression levels of 5 of 17 M. mulatta-specific MDTEs and conducted gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to estimate their functional implications. Further studies are needed, but this research can contribute to the prediction and safety testing of novel therapeutics in preclinical trials by providing the first report of MDTEs that are exclusively present in the M. mulatta genome.In this study, we confirmed MDTEs within the genome of us study . As a reM. mulatta miRNAs, the mml.gff3 file was downloaded from the miRBase v22 database (https://mirbase.org/ (accessed on 9 March 2023)) and a total of 990 mature miRNAs were registered. Among them, miRNAs labelled ‘JSUE’, of which the chromosomal location is uncertain, were excluded. Additionally, the multi-copy miRNAs produced from different chromosomes but having the same mature miRNA sequences were counted only once. Considering these two conditions, a total of 907 mature miRNAs remained. Using BedTools [https://genome.ucsc.edu/cgi-bin/hgTables (accessed on 9 March 2023)). Following the definition of MDTEs established in previous studies, we selected mature miRNAs that were completely covered by TE sequences as MDTEs [M. mulatta were selected for further study, all with a read count of more than 100 from the deep sequencing results. The secondary structures and minimum free energy (MFE) of the pre-miRNAs were calculated using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi (accessed on 12 March 2023)).To identify the genomic coordinates of BedTools , the chras MDTEs ,18. SimpM. mulatta were provided by the National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB). Total RNA was isolated using Hybrid-RTM  according to the manufacturer’s instructions. Total RNA was quantified for both concentration and purity using an ND1000 UV-Vis spectrophotometer . Then, the reverse transcription of total RNA was performed using an HB miR Multi Assay KitTM  in accordance with the manufacturer’s recommendations. The conditions for cDNA synthesis were as follows: incubation at 37 °C for 60 min (SYSTEM I) or 50 °C for 60 min (SYSTEM II), followed by incubation at 95 °C for 5 min and holding at 4 °C.Tissue samples from male and female −ΔΔCt method.To examine the relative expression of miRNAs, qPCR was performed using the HB_I Real-Time PCR Master Mix Kit . Specific primers for miRNA amplification were designed and synthesized by HeimBiotek, Inc. Small nuclear RNA (snRNA) U6 was used to normalize the relative miRNA expression, and the same amount of each cDNA sample was used for the experiment. The PCR was performed on a Quantstudio1 system  and the conditions were as follows: hold at 95 °C for 15 min for initial activation, followed by 40 thermal cycles at 95 °C for 10 s and 60 °C for 40 s; standard melting conditions with 90 s at 55 °C and then 5 s each at 1 °C increments between 55 °C and 99 °C. The ramp rate of the last transformation of 60 °C to 95 °C was set at 0.15 °C/s. All samples were analyzed in triplicate and the relative expression data were analyzed using the 2https://www.targetscan.org/vert_80/ (accessed on 7 July 2023)), a web-based tool that computationally predicts miRNA targets. Predicted targets were required to have a total context ++ score of ≤−0.15 to reduce the number of false positives. Afterward, the selected target genes were subjected to GO and KEGG pathway analysis to examine the enrichment analysis using the web tool ShinyGO 0.80 (http://bioinformatics.sdstate.edu/go80/ (accessed on 3 October 2023)) with a false discovery rate (FDR) cut-off of 0.05. All tools were used with the species set to M. mulatta.Target gene prediction for the miRNAs was performed using TargetScan Human 8.0  of the data was plotted on a bar graph.M. mulatta mature miRNAs and intersected them with the RepeatMasker output file containing the genomic coordinates of repeat sequences in the M. mulatta genome. Consequently, based on mature miRNAs, we confirmed the presence of 101 unique MDTEs in the M. mulatta genome. Detailed information on all the MDTEs is provided in M. mulatta miRNAs registered in miRBase (v22), MDTEs accounted for approximately 11.1% of the total miRNAs  are conserved across species. Apart from the three miRNAs  which are not registered in humans but are present in other species such as horses and common marmosets, we found 17 MDTEs that were only identified in M. mulatta according to the miRBase database (v22)  .Since there is no experimental evidence or previous research on these 17 MDTEs, we examined their expression using quantitative polymerase chain reaction (qPCR). To narrow down the candidates for qPCR verification, criteria based on >100 read counts were used. As a result, five miRNAs  were selected for further analysis. Before qPCR, the hairpin structures of the five MDTEs were predicted using pre-miRNA sequences obtained from the miRBase database (v22). All miRNAs exhibited a stem–loop configuration, as shown in M. mulatta and 13 tissues  from a male M. mulatta  from a female  mulatta .Among the MDTEs, mml-miR-7206-3p was not universally expressed across all tissues, and its expression pattern varied between a female and male. mml-miR-7206-3p was not detected in the large intestine of a female, whereas it was absent in the kidney, large intestine, liver, small intestine, and stomach of a male. Notably, the only tissue in which mml-miR-7206-3p was not expressed in either sex was the large intestine. Additionally, its expression was upregulated in female muscle and male cerebrum tissue.M. mulatta. In a male, it exhibited widespread expression except in the bladder and was particularly pronounced in the pancreas and heart. The expression of mml-miR-7174-5p was significantly higher in the liver of female M. mulatta and in the kidney and stomach of male M. mulatta. The expression of mml-miR-7194-5p was significantly high in the lung of female M. mulatta. Additionally, mml-miR-7194-5p was highly expressed in the kidney and stomach of a male, resembling the expression patterns of mml-miR-7174-5p in male.In contrast, other miRNAs  were expressed in all tissues of both sexes, with distinct expression patterns observed between a female and male. The expression of mml-miR-7163-5p was particularly prominent in the kidneys of both sexes. While showing high expression in all male tissues other than the liver, mml-miR-7163-5p displayed lower expression in the female bladder, cerebellum, ovary, and small intestine. The expression of mml-miR-7168-3p was specific to the liver and muscle of a female M. mulatta using TargetScan Human 8.0. The predicted target genes for each miRNA were selected based on the criteria of a total context++ score ≤ −0.15 . However, the putative target genes of two MDTEs, mml-miR-7163-5p and mml-miR-7174-5p, were not significantly involved in the biological pathways that meet the FDR cut-off of 0.05. As a result, no analysis results were obtained for these two MDTEs. The target genes of the remaining three MDTEs were subjected to KEGG pathway analysis and the results are presented in M. mulatta is one of the most widely used non-human primate species in medical and biological research due to its similarities in physiology and organ function to humans [M. mulatta and humans. Many miRNA gene families are evolutionarily conserved among mammalian species. It is considered that the same conserved miRNAs regulate similar pathways and biological processes among closely related species, such as primates [DNAJB4 and SASH1, in non-muscle-invasive bladder cancer (NMIBC).o humans ,26,27. Tprimates ,35. Howeprimates . After cprimates ,37. Therprimates . Among tM. mulatta and found 17 MDTEs that were only identified in M. mulatta. Among these 17 MDTEs, we conducted GO and KEGG pathway enrichment analysis of five MDTEs’ target genes to gain insights into their associated biological processes. From the KEGG pathway enrichment analysis, mml-miR-7168-3p and mml-miR-7194-5p were involved in various pathways related to diseases such as cancer, type II diabetes mellitus, and viral infection. mml-miR-7206-3p is associated with the innate immune system, the Th17 cell differentiation pathway. For the remaining two miRNAs, mml-miR-7163-5p and mml-miR-7174-5p, no significant enrichment was found from each of their target genes with the FDR cut-off of 0.05. However, with the FDR cut-off of 0.054, mml-miR-7163-5p showed a significant enrichment pathway related to nucleocytoplasmic transport. mml-miR-7174-5p, with the FDR cut-off of 0.051, showed three pathways including SNARE interactions in vesicular transport, adrenergic signaling in cardiomyocytes, and metabolic pathways (In this study, we extended our research on MDTEs to pathways . The assM. mulatta-specific MDTEs that we identified may also exist in other species. Nonetheless, despite these limitations, our study is important because it is the first to classify MDTEs in the M. mulatta genome and to provide experimental data for these MDTEs, the existence of which has been previously unproven. Further studies should be conducted to discern the specific functions of these MDTEs within M. mulatta, and investigating their regulatory roles would provide a better understanding of drug development for human clinical trials.Our study has some limitations as we identified MDTEs based on the genomic coordinates of miRNAs and TEs downloaded from the miRBase database and the UCSC table browser, respectively. With advances in sequencing technologies, many novel miRNAs have been identified in various species. However, our data are limited to miRNAs that are registered only in the miRBase database, and we cannot completely exclude the possibility that the 17


As a sensitive electroactive material, the as-fabricated bimetallic NiCo-MOF nanosheets were employed to modify a screen-printed graphite electrode surface (NiCo-MOF/SPGE) for epinine electro-oxidation. According to the findings, there was a great improvement in the current responses of the epinine because of the appreciable electron transfer reaction and catalytic performance of the as-produced NiCo-MOF nanosheets. Differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry were utilized to analyze the electrochemical activity of the epinine on the NiCo-MOF/SPGE. A linear calibration plot was obtained in the broad concentration range (0.07–335.0 µM) with a high sensitivity (0.1173 µA/µM) and a commendable correlation coefficient (0.9997). The limit of detection (S/N = 3) was estimated at 0.02 µM for the epinine. According to findings from DPV, the electrochemical sensor of the NiCo-MOF/SPGE could co-detect epinine and venlafaxine. The repeatability, reproducibility and stability of the NiCo-metal–organic-framework-nanosheets-modified electrode were investigated, and the relative standard deviations obtained indicated that the NiCo-MOF/SPGE had superior repeatability, reproducibility and stability. The as-constructed sensor was successfully applicable in sensing the study analytes in real specimens.We constructed two-dimensional NiCo-metal–organic-framework (NiCo-MOF) nanosheets based on a facile protocol and then characterized them using multiple approaches (X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), field emission-scanning electron microscopy (FE-SEM), and N N-methyldopamine, deoxyepinephrine or epinine has catecholamine architecture and serves as a natural organic drug. It is present in animals, plants and insects, a with pharmacological profile identical to that of epinephrine. This medicine is a product of the enzymatic hydrolysis of ibopamine, which acts as a cardiovascular drug in the treatment of congestive heart failure , cobalt nitrate hexahydrate [Co(NO3)2·6H2O], 2-Aminoterephthalic acid (NH2-H2BDC) and poly(vinylpyrrolidone) (PVP) were obtained from Sigma-Aldrich . The absolute ethanol and N,N-Dimethylformamide (DMF) came from Merk . Venlafaxine, epinine and all other reagents were of an analytical grade (from Sigma-Aldrich). Orthophosphoric acid was used to prepare the phosphate buffer solution , as a supporting electrolyte.Nickel nitrate hexahydrate [Ni(NO3)2·6H2O  and Ni (NO3)2·6H2O  and were dissolved in water (20 mL). Then, ethanol (20 mL) and DMF (20 mL) were poured into PVP (250 mg) plus NH2-H2BDC (75 mg). The two mixtures were stirred for 30 min to achieve homogeneous and transparent solutions, and were mixed together and stirred in the same way. After 30 min, the obtained solution was placed in a 100-mL Teflon-lined stainless-steel high-pressure autoclave. Subsequently, the autoclave was positioned in an oven for 20 h at 150 °C. Following the reaction, a centrifugation was performed for the appeared product at 10,000 rpm for 15 min using ethanol and DMF to clear the supernatant. The drying of obtained product was performed at 60 °C to collect a purple powder.To this end, Co 6 at diverse scan rates. Using the Randles−Sevcik formula, in the NiCo-MOF/SPGE, the electrode surface was found to be 0.122 cm2, which was approximately 3.9 times greater than that of the unmodified electrode.The surface areas of the NiCo-MOF/SPGE and the unmodified electrode were obtained by CV using 1.0 mM of KThree tablets of the venlafaxine  purchased from a local pharmacy in Kerman (Iran) were finely powdered in a mortar with a pestle. Then, an accurate weighed amount of the homogenized vanlafaxine powder was transferred into a 100 mL 0.1 mol/L PBS (pH 7.0). The contents of the flask were sonicated for 15 min to affect a complete dissolution. Finally, the solutions were filtered and suitable aliquots of the clear filtrate were collected. Afterward, we poured a determined volume of the transparent filtrate into the electrochemical cell consisting of 25 mL of 0.1 mol/L PBS (pH = 7.0) to record the differential pulse voltammograms.Tap water was also sampled, filtrated with a membrane filter and poured into 0.1 M PBS (pH = 7.0). At last, the venlafaxine and epinine contents were measured in the water specimens using the as-developed protocol according to the standard addition method.A facile protocol was followed to construct bimetallic MOFs with great conductivity for developing electrochemical sensors. The conductive NiCo-MOF nanosheets were applied as an effective electrode material for the modification of the SPGE surface to electrochemically co-detect epinine and venlafaxine in real specimens. The commendable features of the NiCo-MOF nanosheets, based on their surface area, porosity and nanoscale dimension, presented a significant reduction in overvoltage and enhancement in the electrochemical behavior of epinine. A robust current was seen for the as-developed modified electrode in response to the epinine oxidation under the optimized circumstances. According to the findings, the linear dynamic range was as broad as 0.07–335.0 μM, and the limit of detection was as narrow as 0.02 μM in the epinine determination. One of the efficient ways to co-detect the two analytes was found to be the complete resolution of the differential pulse voltammetry peak potentials for venlafaxine and epinine . The repeatability, reproducibility and stability of the NiCo-metal–organic-framework-nanosheets-modified electrode were investigated, and the relative standard deviations obtained indicated that the NiCo-MOF/SPGE had superior repeatability, reproducibility and stability. Due to its high reliability and rapid determination of epinine and venlafaxine, NiCo-MOF/SPGE is suggested as a potent electrode for sensing these two drugs in real pharmaceutical matrices and tap water samples.


Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients.  This study aimed to describe the kinetics of  Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up.P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six.  A significant reduction in the parasite load was observed after treatment ( We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases. Leishmania. In the Indian subcontinent, Asia, and Africa, VL is caused by Leishmania donovani and is an anthroponotic disease Leishmania infantum, which is mainly transmitted through the bites of female phlebotomine sand flies of the species Lutzomyia longipalpisVisceral leishmaniasis (VL) is a chronic, neglected disease caused by intracellular protozoa belonging to the genus Relapse is a new VL episode that occurs after an initial cure, usually within 12 months of the initial treatment-Leishmania kinetoplast DNA (kDNA) levels in peripheral blood at the time of diagnosis and during follow-up to evaluate this method for monitoring treatment relapses.One of the main challenges in the treatment of VL is the monitoring and prediction of relapse. Quantitative polymerase chain reaction (qPCR) using peripheral blood samples is an important tool for monitoring treatment responseThe study was conducted in a high-complexity hospital located in the city of Petrolina, Pernambuco, Brazil . The patients were followed-up during the infectious disease service of the municipality.Leishmania positivity in dogsPetrolina is located in the São Francisco Valley, in the Northeast Region of Brazil. It is considered a hyperendemic region for VL, with a mean incidence rate of 4.4 human cases per 100,000 inhabitants between 2007 and 2017The study population consisted of individuals aged ≥ 15 years  who sought health care for a clinical and laboratory condition suspected of VL between July 2017 and July 2020. The cases were confirmed by parasite visualization in bone marrow aspirates. Patients were included in the analysis if they had their blood samples collected before treatment and had at least one follow-up visit after treatment. All patients were tested for human immunodeficiency virus (HIV). Leishmania kDNA using qPCR. In addition, bone marrow samples were collected at baseline for parasite quantification.During follow-up, patients were clinically evaluated at baseline (T0), one day after treatment completion (T1), 3 months (T2), 6 months (T3), and 12 months (T4). Blood samples were collected at each visit for the quantification of This study was approved by the Ethics Committee of the Universidade Federal do Vale do São Francisco (CAAE:68562617.3.0000.5196).After diagnosis, all patients started VL therapy following the recommendations of the Brazilian Ministry of Health. In Brazil, the drugs used for the treatment of VL are N-methyl glucamine antimoniate and amphotericin B. For the choice of medication, age group, pregnancy, and comorbidities are considered. N-methyl glucamine antimoniate was administered at a dose of 20 mg/Sb+5/kg/day, intravenously or intramuscularly, once a day, for 30 days. Liposomal amphotericin B (L-AmB) was intravenously administered at a dose of 4 mg/kg/day for 5 days. Patients with HIV-LV co-infection received secondary prophylaxis with L-AmB at a dose of 4 mg/kg every 15 days. Clinical response was defined as remission of fever, improvement in hematological values, and regression in spleen and/or liver size during therapy. Clinical relapse was defined as the recurrence of VL symptoms and signs within 12 months of treatment.Samples were collected in EDTA tubes  and DNA extraction was performed using the PureLink Genomic DNA Mini Kit  according to the manufacturer’s instructions. The DNA samples were eluted to a final volume of 100 μL. The overall quality of DNA in terms of purity was assessed using a NanoDrop OneC spectrophotometer . DNA samples were frozen at −20 °C until analysis.Leishmania spp. kDNA by qPCR using the primers LEISH-1 (5’-AACTTTTCTGGTCCTCCGG GTAG-3’) and LEISH-2 (5’-ACCCCCAGTTTCCCGCC-3’), as well as the TaqMan-MGB probe (FAM-5’-AAAAATGGGTGCAGAAAT-3’-nonfluorescent quencher-MGB), as described previouslyDNA samples were screened for the presence of L. infantum promastigotes was used as a positive control and also to estimate the parasite load in the patient samples. DNA-free water was used as a non-template control. All qPCR reactions were performed in duplicates. A standard curve prepared using nine serial dilutions  of genomic DNA extracted from cultured P < 0.05.Statistical analyses and graph preparation were performed using GraphPad Prism version 8 . The Kolmogorov-Smirnov test was used to investigate the normality of the data distribution. Statistical differences in median parasite load were determined using the Wilcoxon signed-rank test. Statistical significance was set at During the study period, 38 patients were diagnosed with VL. Four (10.5%) patients died before the end of treatment . Sixteen patients fulfilled the inclusion criteria and were followed-up before (T0) and after treatment (T1). Nine patients were lost to follow-up at T2 (3 months), one at T3 (6 months), and only one who presented with clinical symptoms at T3 returned at T4 (12 months). The baseline clinical and laboratory characteristics of the patients are presented in Leishmania kDNA was detected in all 16 patients with VL at the time of diagnosis (T0), with a median of 2.56 log parasites/mL (range 0.71-4.09). The parasite load in the bone marrow of eight patients with available samples was found to be approximately two times higher than that observed in blood samples at T0 (P = 0.007) (= 0.007) , corroboThirteen patients were treated with L-AmB (81.2%) and three with N-methyl glucamine antimoniate . P < 0.0001) (P = 0.25)  . Two pat = 0.25) .At T2 and T3, the median parasite load was 0.00 and 0.08 log parasites/mL, respectively. Only patient number 11 had an increased parasite load at T2, but had no noticeable clinical signs or symptoms at the time of the visit. At T3, the patient returned to the clinic presenting with asthenia, weight loss, hepatomegaly, and worsening hematological parameters. The patient was treated again with L-AmB. At T4, after the second treatment, the patient no longer had symptoms and there was a reduction in the parasite load . The cliLeishmania kDNA detection by qPCR in blood samples may be a potential target in the diagnosis and post-treatment follow-up of patients in areas endemic for VL.The present study demonstrates that Leishmania kDNA levels at T1. At T2 (3 months), six of seven patients (85.7%) had undetectable Leishmania kDNA levels. Previous studies have demonstrated the usefulness of qPCR in estimating the parasite load in blood samples of VL patients and also observed that despite the significant reduction in parasite load at the end of treatment, it remains detectable in most patients-,We observed a significant reduction in the parasite load immediately after the end of treatment. Despite this reduction, only two patients (12.5%) had undetectable Leishmania DNA levels. Mary et al. (2004)®, which remained at less than 1 parasite/mL. In addition, Aoun et al. (2009)Bossolasco et al. (2003)Leishmania kDNA levels immediately after the completion of treatment. Despite the methodological differences between the studies , the treatment regimen may partly explain the differences in clearance time between the studies, as treatment with L-AmB is administered for 5 days, and treatment with meglumine antimoniate can range between 20 and 40 days. In this study, two of three (66.6%) patients treated with meglumine antimoniate had undetectable L. donovani parasitemia within 30 days of treatment with L-AmB in individuals who relapsed, corroborating other studies that observed an increase above 10 parasites/mL in blood before clinical relapse,In our study, a 67-year-old patient showed a 2-log increase in parasite load 3 months after treatment, and relapsed clinically at month six. Advanced age has been associated with relapse in immunocompetent patients treated with L-AmBL. infantum using L-AmB. This study presents data on the detection of Leishmania kDNA using qPCR in a series of cases followed-up longitudinally after therapy. One case of clinical relapse presented an increased parasite load before symptom onset. Further studies with larger populations are needed to evaluate the use of this technique, as well as the definition of a cutoff point, in the prediction of relapse in VL patients.This study had some limitations, including the small sample size and significant loss to follow-up. However, few studies have evaluated the role of therapeutic monitoring by qPCR in the blood of individuals infected with


For external validation, there was a significant correlation between the predicted and observed half lives in the validation cohort. The median half life deviation was +1.53 h, +1.28 h, and +1.79 h for the equations of non-O group, O group, and overall group patients, respectively. In total, eight predictors influencing rFVIII-Fc half life were identified. Prediction equations of rFVIII-Fc half life were developed for the non-O and O blood groups and overall PwHA with a good degree of external validation. The equations could be applied to patients aged 8–64 without the need for PK blood sampling and clinically valuable for personalized therapy.The half life of recombinant factor VIII-Fc (rFVIII-Fc) for people with hemophilia A (PwHA) varies greatly. Understanding the factors influencing the variation and assessment of rFVIII-Fc half life is important for personalized treatment. Eighty-five severe-type PwHA with rFVIII-Fc treatment receiving an evaluation of half life by the Web-Accessible Population Pharmacokinetic (PK) Service—Hemophilia during 2019–2021 were retrospectively enrolled. The 50-patient PK profiles before 2021 were used for analysis and developing prediction models of half life, and the 35-patient PK profiles in 2021 were used for external validation. The patients in the development cohort were aged 8–64, with a median rFVIII-Fc half life of 20.75 h . By multivariate linear regression analysis, we found two, four, and five predictors of rFVIII-Fc half life for the blood groups non-O, O patients, and overall patients, respectively, including baseline VWF:Ag, BMI, VWF:activity/VWF:Ag ratio, body weight, O blood group, inhibitor history, HCV infection, and hematocrit. The three prediction equations of rFVIII-Fc half life (T) were respectively developed as T for non-O group patients = −0.81 + 0.63 × (BMI, kg/m Hemophilia A is the second-most common inherited bleeding disorder and only less frequent than von Willebrand disease (VWD). The common symptoms in people with hemophilia A (PwHA) include joint and muscle bleeding, and repeated joint bleeding could damage joints even in early life. Regular prophylaxis can reduce the joint bleeding rate and promote joint protection, and it has been the standard therapy for severe-type PwHA since 2007 ,2,3. HowInter-individual variation in the half life of SHL FVIII was reported to be evident and related to the pre-infused von Willebrand factor (VWF) level ,14. VariAlthough rFVIII-Fc half life can be assessed by the Web-Accessible Population Pharmacokinetic Service—Hemophilia (WAPPS-Hemo), it still requires at least two-point blood sampling, which requires specific sampling intervals, a lead time for reports of FVIII:C levels, and a waiting time for an answer from the WAPPS-Hemo ,29. For The primary goal of our study was to search for potential predictors of rFVIII-Fc half life from the covariates of real-world clinical practice. The secondary goal was to develop prediction models of rFVIII-Fc half life with external validation that could be clinically more convenient when individualizing regimens for PwHA on rFVIII-Fc.rFVIII-Fc was launched in Taiwan in November, 2018. After that, many previously treated PwHA who had been registered at Taipei Medical University Hospital (TMUH) and Tri-Service General Hospital (TSGH)—the two tertiary referral centers for PwHA—switched in succession from SHL rFVIII, including Kogenate-FS, Advate, Xyntha, and Kovaltry, to rFVIII-Fc between 2019 and 2021. Because it was the first time that Taiwanese patients began to use EHL products and because there were no Taiwanese PwHA participating in the clinical trials on rFVIII-Fc, the patients who switched to rFVIII-Fc, at that time, were encouraged to receive a PK assessment by the WAPPS-Hemo. The data on rFVIII-Fc half lives that were extracted from the WAPPS-Hemo were recorded for clinical practice.The inclusion criteria included people with severe hemophilia A who had received rFVIII-Fc treatment, no matter whether it was on-demand therapy or prophylaxis therapy, and who had received a PK assessment by the WAPPS-Hemo. The exclusion criteria included people with non-severe (moderate and mild) hemophilia A, patients without a PK assessment, patients with current FVIII inhibitors, and patients with a combination of other bleeding disorders, such as VWD. A total of 85 people with severe hemophilia A were eligible and included in the analysis.The overall cohort of 85 PwHA could be simply divided into two cohorts. Among the 85 patients, there were 50 whose PK data from the WAPPS-hemo were obtained before the end of 2020 and who are referred to as cohort 2019–2020. Regarding the other 35 PwHA who switched to rFVIII-Fc much later, their PK data were obtained in 2021 and we refer to them as cohort 2021. For the purposes of the study, the data on cohort 2019–2020 were used as the development cohort for predictor analysis and the development of prediction models, and the data on cohort 2021 were used as the validation cohort for the external validation of the predictive models. Since hemophilia A is a rare disorder and the number of cases is relatively limited, and since this is a retrospective study, in which it was hard to know ahead of time whether there was an adequate case number, an analysis of the sample size necessary for the development of the study, which is a commonly used method in many prospective studies, was not carried out prior to the study. Therefore, a posteriori analysis would be of interest to assess the power of the results obtained. We used external validation to examine the prediction models developed and assess their applicability by analyzing the median deviation between predicted and observed values. This retrospective observational cohort study was approved by the ethics review board of both institutions (IRB number: A202105193 for TSGH and N202111052 for TMUH).The charts of all 85 PwHA were reviewed. Clinical data were collected for analysis, including age, body mass index (BMI), body weight (BW), ABO blood groups, hematocrit (Hct), baseline VWF:Ag and VWF:activity, and rFVIII-Fc half lives by the WAPPS-Hemo. VWF:activity means the data obtained via VWF:ACL activity or VWF:RCo. The VWF:activity/VWF:Ag ratio was also used as one of the clinical variables because this ratio can represent the quality or functioning ratio of all the VWF molecules and consequently influence FVIII in PwHA. Inhibitor history was defined as PwHA who had had a transient low-titer inhibitor or high-responder inhibitors eradicated by immune tolerance induction (ITI) therapy before the study period. HCV infection was defined as PwHA with positive anti-HCV antibodies. HIV infection was defined as PwHA with positive anti-HIV antibodies and a PCR for HIV.Among the 85 PwHA who had a PK assessment, 83 received their PK assessment during regular prophylaxis (RP) with a regimen of 55–65 iu/kg every 5 days, 45–50 iu/kg every 4 days, 30–35 iu/kg every 3 days, 35–50 iu/kg twice weekly , and 65 iu/kg once weekly, which was performed according to the regulations for the payments of the National Health Insurance program. When the PK assessment was performed, all patients were in a stable condition without recent bleeding. They received at least 2-spot blood sampling on the following spots: trough-level and, additionally, any two or three spots of post-infusion . However, data on FVIII:C levels < 1% were abandoned in the calculation of half lives. The blood sampling numbers and times for a PK assessment depend on the convenience of the patient and the clinical physician’s decision. Therefore, some patients received 2-spot sampling while the others received 3-spot or 4-spot sampling. All FVIII:C levels were checked by one-stage assay. All rFVIII-Fc half lives were calculated by the WAPPS-Hemo. The remaining 2 patients received on-demand therapy with rFVIII-Fc and they also received PK assessments with 2-spot blood sampling, including trough-level and post-infusion (12 h). All data on baseline VWF levels were obtained in a stable condition during regular comprehensive care or before rFVIII-Fc infusion when receiving a PK assessment. There were 4 patients whose data on VWF levels were unavailable.Clinical variables were analyzed in order to search for potential predictors. By stepwise backward linear regression, independent predictors from clinical covariates could be found. By multivariate linear regression analysis, we attempted to develop prediction models; when successful, internal validation with the development cohort was performed by examining the median deviation from the values of the predicted rFVIII-Fc half life minus the observed rFVIII-Fc half life and by correlation analysis between predicted half life and observed half life values.The data on the validation cohort in 2021 were used for external validation of the prediction models established by the development cohort. Additionally, the median deviation from the values of the predicted rFVIII-Fc half life minus the observed rFVIII-Fc half life was recorded for analysis. Correlation analysis between predicted rFVIII-Fc half life and observed rFVIII-Fc half life values was also performed to examine the predictive performance.t test, the Mann–Whitney test, a Chi square test, and Fisher’s exact test. After that, we studied the relationship between half life and baseline VWF:Ag, baseline VWF:activity, BMI, and BW by Pearson’s correlation analysis. Because many of the covariates interacted with each other, in order to investigate the interdependence of these variables and determine which variables were independent factors affecting rFVIII-Fc half life, we used univariate linear regression (LR) analysis and stepwise-backwards multivariate LR analysis to search for independent predictors affecting FVIII PK. For this, we used SAS and R software version 2.12.0 . A p-value < 0.05 was considered statistically significant.We analyzed patient data on clinical characteristics by an independent p < 0.01 **) and a higher VWF:activity/VWF:Ag ratio . Regarding rFVIII-Fc half life, we found a large amount of variation in both the development cohort and the validation cohort .A comparison of the clinical features of the 50 PwHA in the development cohort and the 35 PwHA in the validation cohort is shown in When we examined the rFVIII-Fc half lives in the development cohort, a large amount of inter-patient variation was found as shown in p < 0.05 *). The mean half lives of patients with a baseline VWF:activity level of <100% and ≥100% were 17.07 h and 24.63 h, respectively (p < 0.01 **). However, VWF:activity/VWF:Ag ratio , Hct , and HIV infection were not significantly associated with half life.The results of the analysis of patient characteristics and half lives in the development cohort by the Mann–Whitney test are shown in p < 0.01 **). The rFVIII-Fc half lives of non-O group and O group patients were 22.83 ± 6.46 h and 16.26 ± 4.61 h, respectively (p < 0.001 ***).When comparing the characteristics of non-O blood group PwHA and O blood group PwHA, we found no significant difference in age, BMI, BW, Hct, inhibitor history, HIV infection, or HCV infection between the two groups. However, the baseline VWF:Ag level of non-O group and O group patients was 132.1 ± 63.7% and 86.9 ± 19.3%, respectively (p < 0.0001 ***). BW and BMI were weak positive correlates to half life , and both baseline VWF:Ag and baseline VWF:activity were strong positive correlates to half life . The results of the analysis are shown in p < 0.05 * and Pearson-rank = 0.5320, p < 0.01 **, respectively). BMI was found to have a weak positive correlation to both baseline VWF:Ag and baseline VWF:activity . Given the different slopes in the relationship with rFVIII-Fc half life between blood group O and blood group non-O patients, as well as the higher number of cases with a mix of blood group O and blood group non-O patients, the relationship between the various covariates and rFVIII-Fc half life were analyzed separately for blood group O and blood group non-O and together for the overall development cohort.By Pearson’s correlation analysis of rFVIII-Fc half life in the development cohort, we found that age was a moderate positive correlate to half life When analyzing non-O blood group PwHA using this method, age, BW, Hct, inhibitor history, HCV infection, HIV infection, baseline VWF:activity, and the VWF:activity/VWF:Ag ratio were eliminated as predictive parameters. The remaining covariates (baseline VWF:Ag and BMI) were proved to be positive predictors and were used to define Equation (1), which explained 51.97% of all the variability in the rFVIII-Fc half life. As per the mathematical modeling, the rFVIII-Fc half life would increase by 6.07 h whenever the baseline VWF:Ag is increased by 1 IU/mL (100%).(2)When analyzing O blood group PwHA using this method, age, Hct, inhibitor history, HCV infection, HIV infection, and baseline VWF:activity were eliminated as predictive parameters. The remaining covariates  were proved to be predictors and were used to define Equation (2), which explained 75.17% of all the variability in the rFVIII-Fc half life. As per the mathematical modeling, the rFVIII-Fc half life would increase by at least (for it still needs to be added to the impact of the VWF:activity/VWF:Ag ratio) 13.3 h whenever the baseline VWF:Ag is increased by 1 IU/mL (100%).(3)When analyzing the overall cohort of PwHA (n = 46) using this method, age, BW, BMI, HIV infection, baseline VWF:activity, and the VWF:activity/VWF:Ag ratio were eliminated as predictive parameters. The remaining covariates  were proved to be predictors and were used to define Equation (3), which explained 66.38% of all the variability in the rFVIII-Fc half-life. As per the mathematical modeling, the rFVIII-Fc half life would increase by 7.24 h whenever the baseline VWF:Ag is increased by 1 IU/mL (100%), decrease by 3.84 h in patients with a positive inhibitor history, increase by 2.99 h in patients with a positive HCV infection test, and decrease by 2.83 h in patients in blood group O.T  = −1.76 + 7.24 ×  − 3.84 × (Inhibitor history) + 2.99 × (HCV infection) − 2.83 × (O blood group) + 0.30 × (3)Regarding the model performance of the prediction equations, differences in the observed and predicted rFVIII-Fc half life values in the prediction models were examined with the data of the development cohort. The median deviation was +0.27 h for Equation (1) of blood group non-O PwHA, −0.04 h for Equation (2) of blood group O PwHA, and +0.79 h for Equation (3) of the overall development cohort, with a standard deviation for the difference of ±4.53 h, ±2.03 h, and ±3.78 h, respectively. Thus, there were a slight tendency to overestimate the rFVIII-Fc half life using Equations (1) and (3) but a slight tendency to underestimate it using Equation (2). When comparing the predicted and observed half lives, more than 80% of the predicted half lives for blood group non-O patients, blood group O patients, and the overall cohort were within a 4.6 h, 2.24 h, and 4.2 h difference of the observed half life, respectively. The predicted half life was within a 4 h difference of the observed half life in 19 (73.1%) of the 26 blood group non-O PwHA by Equation (1), in 19 (95%) of the 20 blood group-O PwHA by Equation (2), and in 33 (71.7%) of the 46 PwHA by Equation (3). The correlation between the predicted and observed rFVIII-Fc half life values is shown in In order to examine the predictive performance of the three models, the data on the validation cohort were used for external validation. As calculated by the three prediction equations, the median half life deviation was +1.53 h for Equation (1) of blood group non-O PwHA, +1.28 h for Equation (2) of blood group O PwHA, and +1.79 h for Equation (3) of the overall PwHA cohort, with a standard deviation for the difference of ±6.04 h, ±3.63 h, and ±4.48 h, respectively. Thus, there was a slight tendency to overestimate the rFVIII-Fc half life using Equations (1)–(3). The correlation between the predicted and observed rFVIII-Fc half life values is shown in In the analysis of our cohort, the real-world data on the rFVIII-Fc half life show a large amount of inter-patient variation  compared with the relatively homogeneous data on the rFVIII-Fc half life in the A-long study  . Understp < 0.001 ***). Moreover, in the univariate LR analysis, age was also significantly associated with rFVIII-Fc half life in blood group non-O patients, blood group O patients, and the overall development cohort. These findings are compatible with previous reports that the half life of FVIII and rFVIII-Fc in children with hemophilia A is shorter than that in adult patients [In our study, those whose age was <20 and those whose age was ≥20 had different mean half lives . This is much longer than the difference in the SHL rFVIII half life in the report of Fischer [p < 0.01 **), which is similar to previous reports in which VWF levels were reported to be 25% to nearly 50% higher in healthy persons in the non-O blood group [Many authors have found that the O blood group can impact the FVIII half life dependently due to O blood group patients having a lower VWF level and a lower FVIII level ,18,19,20 Fischer  (nearly  Fischer  (2.6 h). Fischer . In our od group ,42. The od group . Dino MeTo develop the prediction models, we used stepwise-backwards MVLR analysis, which has been shown to be a better approach than stepwise-forward MVLR analysis by some authors ,44. Becap-value became more significant and the P-rank of the correlation became stronger. Because hemophilia A is a rare disease with a prevalence of 1/10,000 and the total number of cases in our cohort was relatively small, the case number that could be used for external validation  was quite limited, especially for the non-O and O blood group patients whose case numbers were even smaller . Therefore, despite the good R2 values of the predictive models for non-O and O blood group patients , the P-rank of the correlation between the observed and predicted half lives indicated a moderate, not strong, correlation, which may be due to overfitting. However, despite the small number of cases for external validation, the trend of the results showed that the three prediction models can be applied in and are feasible for clinical practice.Before prediction models are adopted in clinical practice, both internal validation and external validation are necessary . We used2 values of the three predictive models are sufficient for the non-O group (51.97%), O group (75.17%), and overall cohort (65.38%) models. However, it was also found that these predictors did not completely describe the within-group variability. There may be other covariates that have not been put into the linear regression analysis. For example, the level of activity with the possibility of subclinical bleeding that we previously mentioned [Regarding the clinical applications of our results, any severe-type PwHA on rFVIII-Fc treatment could have their half life calculated through two of the three prediction equations . A clinical physician could therefore obtain the two predictive rFVIII-Fc half life values for the prescribed rFVIII-Fc regimen. The Rentioned ,31 and bentioned  may be i2 values.The limitations of our study are as follows. First, our sample size was relatively small for both the development cohort and the validation cohort. This is related to the fact that hemophilia A is a rare disease, rFVIII-Fc is one of several EHL rFVIII products used in hemophilia treatment centers in Taiwan, and other EHL products were not included in the study. Second, all of the observed rFVIII-Fc half life values used in this study were calculated by the population PK model in the WAPPS-hem and not by a classical PK study for FVIII from 11 samples as per the recommendations from the International Society on Thrombosis and Hemostasis . Third, In conclusion, we identified eight predictors impacting rFVIII-Fc half life from real-world data, including baseline VWF:Ag, O blood group, inhibitor history, HCV infection, Hct, BMI, VWF:activity/VWF:Ag ratio, and BW. Moreover, we developed three prediction models for O group patients, non-O group patients, and the overall cohort that explained 51~75% of the rFVIII-Fc half life variability and examined their predictive performance through internal and external validation. The prediction models could be clinically applied to PwHA on rFVIII-Fc treatment for the purpose of routine regimen adjustments without blood sampling of FVIII:C levels. We believe that our results are of value to personalized therapy for hemophilia patients and provide insight into predictors of the rFVIII-Fc half life. Regarding the mechanism underlying how these predictors influence the rFVIII-Fc half life, further study is needed in order to clarify the cause–effect relationship and the interactions among these predictors.


It has been proposed to implement the >100 Gb/s data-center interconnects using a two-channel optical time-division multiplexed system with multilevel pulse-amplitude modulation. Unlike the conventional four-channel optical time-division multiplexed system which requires an expensive narrow pulse, the two-channel system can be implemented cost-effectively using a wide pulse (which can be simply generated using a single modulator). The two-channel system is expected to be practically available using an integrated transmitter in a chip due to the recent advances in photonics-integrated circuits. This paper reviews the current stage of research on a two-channel optical time-division multiplexed system and discusses possible research directions. Furthermore, it has been demonstrated that 200 Gb/s signals can be generated by using modulators with only 17.2 GHz bandwidth. Therefore, the use of the phase-alternating pulse can make the multiplexed signal robust to chromatic dispersion, enabling the 200 Gb/s 4-level pulse-amplitude-modulated signal to be transmitted over 1.9 km of standard single-mode fiber. Data centers have become essential infrastructure for society due to the rapid exploration of data traffic through high-definition video and audio streaming services, cloud services, and social networks ,2,3,4. Tstandard ,6. Consistandard ,8, it isals soon ,10. Sincals soon , variousals soon ,13. Howeals soon . Thus, Pals soon , a machials soon , or maxials soon , to imprals soon ,17,18. TJ. Verbist et al. proposed using optical time-division multiplexing (OTDM) to increase the operating speed of DCIs . They deN signal [Recently, we proposed the implementation of high-speed DCIs with a two-channel OTDM-PAM-N signal . In thisN signal ,28. The This paper reviews recent research on two-channel OTDM systems. The remainder of the paper is organized as follows: B is necessary to generate a two-channel OTDM signal with a baud rate of B can be generated using a Mach–Zehnder modulator (MZM) (biased at the quadrature point) with an electrical clock at a frequency of B, as illustrated in An optical pulse with a repetition rate of 3 MZM1 (3-dB bandwidth = 17.2 GHz). The MZM was biased at the quadrature point and driven by a 25 GHz electrical clock. The pulse with a 7.7-dB ER was modulated using an MZM2 to generate the 75 Gb/s RZ-PAM-8 signal. The electrical PAM-8 signal was generated by a 3-bit DAC and three outputs from a pulse-pattern generator (PPG). The MZM2 was driven through a push–pull operation using 75 Gb/s differential pair signals from the DAC. Instead of SOAs, erbium-doped fiber amplifiers (EDFAs) were used to compensate for the insertion losses of MZMs. The RZ signal was split into two parts using a polarization beam splitter (PBS) and combined again using a polarization beam combiner (PBC). A proper delay line was inserted in one of the paths to realize decorrelation and accurate timing alignment (for multiplexing). The generated signal was transmitted over a 1.9 km standard single-mode fiber (SSMF) and detected by a receiver consisting of a PIN PD (3 dB bandwidth = 70 GHz) and an electrical amplifier (3-dB bandwidth = 50 GHz). After the signal was captured using a real-time oscilloscope , it was demultiplexed and equalized using a N . T’ represents the transpose. A multilayer equalizer for the two-channel OTDM system was proposed to reduce the BER floor , as shown in ectively . h1 and r-th order filter coefficient, and k-th input and output of the equalizer, respectively [While estimating the optimal sampling point using the FFE-based demultiplexer, the BER can be further reduced using an improved MIMO equalizer. The MIMO equalizer may consist of second-order Volterra equalizers, where the input–output relationship can be expressed as follows: ectively . A paramUsing the data received from the setup shown in B to suppress the carrier frequency  and a periodic optical filter . When thAs illustrated in The transmission performance of the 200 Gb/s OTDM signal generated using the CSRZ pulse  was compared with that generated using the RZ pulse . The 1549.9 nm light was phase-modulated with a 25 GHz electrical clock  chip, as shown in The optical pulse source with a high repetition rate is a key component of the two-channel OTDM system. A technique wherein an MZM is biased at a sub-quadrature point was proposed to increase the ER of the optical pulse with a high repetition rate . This teics chip . Since tics chip , it is nN signal) due to the nonlinear transfer characteristics arising from gain saturation [The high insertion loss of the transmitter is one of the disadvantages of the system. Although EDFAs were used in the previous experiments ,30,34, wturation ,37. The As described in With a multilevel PAM modulation format, it is anticipated that 800 gigabit and 1.6 terabit Ethernet will be deployed soon. Considering the SINAD, the modulation level of PAM would not exceed eight. To further increase the line speed despite the limitation, a two-channel OTDM system was proposed, which is reviewed in this paper. In the system, the two-channel multiplexed signals are generated using a sinusoidal pulse and detected using a single PD. Compared to the four-channel OTDM system, the optical pulse can be simply generated using a modulator. We believe that this system is commercially deployable by integrating the transmitter into a single chip. Research has demonstrated that a transmitter consisting of three 17.2 GHz MZMs could generate the 150 Gb/s OTDM-PAM-8 signal when the FEC limit was set to


Researchers in the field of tissue engineering are always searching for new scaffolds for bone repair. Polyetheretherketone (PEEK) is a chemically inert polymer that is insoluble in conventional solvents. PEEK’s great potential in tissue engineering applications arises from its ability to not induce adverse reactions when in contact with biological tissues and its mechanical properties, which are similar to those of human bone. These exceptional features are limited by the bio-inertness of PEEK, which causes poor osteogenesis on the implant surface. Here, we demonstrated that the covalent grafting of the sequence (48–69) mapped on the BMP-2 growth factor (GBMP1α) significantly enhances the mineralization and gene expression of human osteoblasts. Different chemical methods were employed for covalently grafting the peptide onto 3D-printed PEEK disks: (a) the reaction between PEEK carbonyls and amino-oxy groups inserted in the peptides’ N-terminal sites (oxime chemistry) and (b) the photoactivation of azido groups present in the peptides’ N-terminal sites, which produces nitrene radicals able to react with PEEK surface. The peptide-induced PEEK surface modification was assessed using X-ray photoelectron measurements, while the superficial properties of the functionalized material were analyzed by means of atomic force microscopy and force spectroscopy. Live and dead assays and SEM measurements showed greater cell cover on functionalized samples than the control, without any cytotoxicity induction. Moreover, functionalization improved the rate of cell proliferation and the amount of calcium deposits, as demonstrated by the AlamarBlue™ and alizarin red results, respectively. The effects of GBMP1α on h-osteoblast gene expression were assayed using quantitative real-time polymerase chain reaction. The increased life expectancy in Western countries has given rise to higher demands for orthopedic implants in order to address bone and cartilage pathologies . TraditiThe scientific Interest in PEEK is increasing as a result of its potential use in 3D printing, since it is a thermoplastic polymer that can be shaped in its melting temperature range ,10, arouPEEK has shown high resistance to thermal degradation, organic and aqueous environment-related degradation, and biodegradation . ConsequIn the literature, several physical methods for the surface modification of PEEK have been proposed: thermal spraying , pulsed Previous studies have proposed two different methods for PEEK functionalization. In the work of Yakufu et al., cell adhesion or proliferation were promoted by anchoring signal sequences to the isocyanate or amino groups, previously introduced through the reduction of the carbonyl groups in PEEK into alcohol groups ,21,22. AMerck Millipore  provided acetic acid, acetone, acetonitrile, Fmoc-protected amino acids, Bis-Boc-aminooxy acetic acid (Aoa), dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), ethyl cyano(hydroxyimino)acetate (Oxyma Pure), methanol, Rink amide MBHA resin, triethylamine (TEA), triethylsilane (TES), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU). Biosolve  provided diethyl ether and trifluoroacetic acid (TFA). PanReac Appli-Chem  provided 2-propanol for HPLC , while Carlo Erba  provided ethanol. Iris Biotech GmbH  provided N-Methyl-2-pyrrolidone (NMP).α (sequence: H-Aoa-(7-aminoheptanoic acid)- Pro-Phe-Pro-Leu-Ala-Asp-His-Leu-Asn-Ser-Thr-Asn-His-Ala-Ile-Val-Gln-Thr-Leu-Val-Asn-Ser-NH2) was synthesized using solid-phase peptide synthesis (SPPS) on Rink amide MBHA resin with a Syro I synthesizer . The N-termini of all amino acids were protected with an Fmoc group and the side chains of His, Asn, and Gln were protected with Trt, Asp with OtBu, and Ser/Thr with tBu. A double coupling strategy was used for every amino acid addition. Five equivalents of each amino acid, five equivalents of activating agent HBTU/Oxima Pure, and ten equivalents of DIPEA, with respect to the resin reactive groups, were used for each coupling of 45 min. As a spacer (x) between the bioactive peptide sequence and the Aoa, 7-aminoheptanoic acid was introduced with the same procedure. Aoa was added to the sequence through a double coupling with five equivalents, in addition to five equivalents of activating agent HBTU/Oxima Pure and ten equivalents of collidine, with respect to the resin reactive groups, for each coupling of 45 min. After that, the peptide was cleaved from the resin and deprotected from the Bis-Boc of Aoa and the side chain-protecting groups using 4.75 mL TFA, 0.125 mL TES, and 0.125 H2O for 1.5 h at room temperature. The resin was filtered off. The solution was concentrated, and the crude peptide was precipitated with cold diethyl ether addition. The identity of the peptide was determined through mass spectrometry . The peptide was purified using RP-HPLC and characterized through analytical RP-HPLC using a Vydac C18 column  in the following conditions: eluent A (0.05% TFA in MilliQ water); eluent B (0.05% TFA in 40% isopropanol and 60% MilliQ water); gradient, from 60 to 80% B in 40 min; flow rate, 0.5 mL/min; detection at 214 nm (retention time: 24.17 min).The peptide Aoa-x-GBMP13-x-GBMP1α (sequence: H-Phe(p-N3)-Gly-γAbu-Pro-Phe-Pro-Leu-Ala-Asp-His-Leu-Asn-Ser-Thr-Asn-Ser-Thr-Asn-His-Ala-Ile-Val-Gln-Thr-Leu-Val-Asn-Ser-NH2) was synthesized as reported for Aoa-x-GBMP1α synthesis. γAbu and Gly were introduced as a spacer (x) between the bioactive peptide sequence and Aoa . Finally, the sequence was enriched with an azido group on the N-terminus through a double coupling of Fmoc-p-azido-Phe-OH with the same protocol used for the other amino acids. Therefore, the deprotection of the peptide from the Fmoc occurred before the cleavage from the resin and deprotection from the side chain-protecting groups using 4.75 mL TFA, 0.125 mL TES, and 0.125 H2O for 90 min at room temperature. The crude peptide in solution was separated from the resin, concentrated, precipitated with cold di-ethyl ether, and filtered. The peptide mass was investigated using mass spectrometry  to confirm the peptide identity. The peptide was purified using semipreparative RP-HPLC and analyzed through RP-HPLC using a Vydac C18 column  in the following conditions: eluent A (0.05% TFA in MilliQ water); eluent B (0.05% TFA in acetonitrile); gradient, from 27 to 37% B in 20 min; flow rate, 1 mL/min; detection at 214 nm. The peptide’s retention time was 9.874 min.The peptide Nstl format and imported into Simplify 3D software . The files were sliced in order to generate printer-ready G code, which was then printed using an Apium P155 PEEK filament printer . The printer used a PEEK filament  with a diameter of 1.75 mm and extruded it through a 0.4 mm diameter nozzle at a temperature of 485 °C onto a print bed with a temperature of 130 °C. The print speed was set to 33.3 mm/s and the resulting 3D-printed PEEK disks had a flat surface on one side and a patterned surface on the other. The PEEK disks were 10 mm in diameter and 4 mm in height, with a 100% infill. The PEEK disks were designed with a flat (lower side) and a patterned surface  computer-aided design (CAD) software was used to create PEEK disks files, which were then exported in All the peptides were grafted to PEEK disks.−4 M, since [Aoa-x-GBMP1α was bound through chemoselective ligation, whereby the amino-oxy group present on the N-terminus of the peptide chain was linked to carbonyl groups on the PEEK surface. A solution of peptide in 40 mM monobasic sodium phosphate at pH 6 was left to react with PEEK samples for 24 h at room temperature. At the end of the reaction, the samples were washed 3 times with the buffer and 3 times with MilliQ water . The pepM, since  proved t3-x-GBMP1α occurred by dissolving the peptide in MilliQ water and leaving the solution reacting with the PEEK disks. The reaction lasted 3 h at room temperature. Then, the samples were dried out under vacuum in presence of P2O5 for 3 h. A UV lamp irradiated the dry samples for 30 min at 254 nm  [Functionalization with NCA, USA) . The fun3-x-GBMP1α were called PEEK-AoaBMP or PEEK-N3BMP, respectively.Samples functionalized with either Aoa-x-GBMP1α or N−8 Torr before being transferred to the analysis chamber, which maintained a vacuum pressure of 10−8–10−10 Torr during measurements. The XPS investigations were conducted on flat PEEK surfaces; non-monochromatized Mg Kα (1253.6 eV) X-ray radiation was used. All the spectra were calibrated in energy, using the C 1s signal of aromatic–aliphatic carbons located at 284.7 eV binding energy (BE) as reference. After the subtraction of a Shirley-type background, curve fitting analysis of the C1s, N1s, and O1s spectra was carried out using Gaussian curves as fitting functions. The same full width at half maximum (FWHM) value was set for all individual photoemission peaks in a complex spectrum. Atomic ratio values and surface atomic concentrations were determined from peak areas, using Scofield’s atomic sensitivity factors [A custom-built instrument comprising preparation and analysis ultra-high-vacuum (UHV) chambers and previously described in  was used factors .−1, tip radius < 10 nm). The AFM measurements were carried out in triplicate, and the cantilever spring constant was measured using the Sader method [F is the applied force, adF is the adhesion force, Es is the Young’s modulus, sν is the Poisson’s ratio for the sample, R is the radius of the spherical indenter, and δ is the elastic indentation depth. Each surface was indented in different areas and approximately three hundred curves were obtained, with a tip–sample approaching rate of 0.3 µms−1 for all force curves. The DMT model was used to fit the experimental curves in the elastic region and calculate the Young’s modulus. The distribution of the elastic modulus was analyzed using OriginPro 8.5 software. Au-coated silicon nitride tips with spring constants ranging from 0.003 to 0.13 N/m were employed to carry out adhesion force measurements. These tips were calibrated via the Sader method. The adhesion force was determined by measuring the pull-off force required to separate the AFM tip from the surface. The PEEK surfaces were probed in various regions, and approximately three hundred approach–retract cycles were recorded for each system. The tip–sample approaching velocity remained constant at 0.3 μs−1 for all force curves. Hooke’s law was used to calculate the adhesion force between the tip and surface, based on the cantilever deflection distance and cantilever spring constant:FS analysis was conducted on non-functionalized and functionalized PEEK at room temperature to investigate the impact of peptides on the mechanical properties of interfaces. The Young’s modulus was determined from the force–distance curves collected using an NTEGRA Atomic Force Microscope  and stiff single-crystal silicon cantilevers with a symmetric tip shape  were utilized . Prior to seeding, the PEEK specimens were subjected to sterilization in 70% ethanol for 24 h and subsequently washed with PBS. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 mL of 1 M HEPES, 5 mL of 200 mM L-glutamine, 5 mL of 100× penicillin/streptomycin, 50 mL of Fetal Calf Serum (FCS), 5 mL of minimal essential medium, and 0.075 g of solid ascorbate acid. The HOB cells were microseeded at 1 × 105 HOB cells per disk was employed. The culture medium was refreshed every three days to sustain optimal growth conditions.To assess gene expression, a density of 2 × 10TM Viability/Cytotoxicity Kit  was used after 48 h of cell culture. Prior to exposure to the live/dead stain, two PBS washings were carried out. Images of the stained samples were captured with an Olympus IX51  fluorescent microscope, and ImageJ software (version 1.8.0_322) was used to merge the images of living cells (green fluorescence at 530 nm) and dead cells (red fluorescence at 600 nm) of the same location into one image for analysis.In order to evaluate cell viability, a LIVE/DEADThe morphology of HOB cells on the 3D-printed PEEK structures was observed using SEM  at a voltage of 10kV. Samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for at least 24 h at 4 °C, 48 h after seeding, to preserve the cell structure. Afterward, the samples were rinsed with 0.1 M sodium cacodylate buffer solution and treated with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h. The samples were then treated with 1% tannic acid in 0.1 M sodium cacodylate buffer for 1 h. After treatment, the samples were dehydrated in a series of aqueous ethanol solutions , twice for each concentration, with each treatment lasting for 5 min. Following dehydration, the samples were air-dried and sputter-coated with gold before SEM observation.To evaluate calcium deposition in HOB cells, alizarin red S staining was performed. alizarin red S is an anthraquinone dye that reacts with calcium, forming a birefringent alizarin red S/calcium complex. HOB cells were cultured for 21 days, after which the PEEK disks were washed in PBS, fixed in 10% formal saline for 15 min, and washed with deionized water thrice. The specimens were then treated with 2% alizarin red S solution for 15 min, followed by five washes in 50% ethanol.The stained scaffolds were assessed using the protocol developed by Lee et al. . To releTM assay was used to assess the proliferation of osteoblasts over a period of 21 days on functionalized and non-functionalized PEEK disks. The fluorescence values were measured at a test wavelength of 570 nm and a reference wavelength of 630 nm using a microplate reader . Each sample was measured in triplicate to ensure the accuracy of the results. Data were collected at 1, 4, 7, 14, and 21 days to monitor the temporal profile of osteoblast proliferation.The AlamarBlueThe present study aimed to investigate the expression levels of specific mRNA transcripts encoding human SRY-Box transcription factor 9 (SOX9), Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN) in HOB cultured on functionalized and non-functionalized PEEK after 1, 7, 14, and 21 days. Total RNA was extracted from the cells at the end of the incubation period using the SV Total RNA Isolation System kit , and any contaminating DNA was removed through DNase I digestion . The cDNA synthesis and subsequent polymerization were performed in a single step using the iTaq Universal SYBR Green One-Step Kit , with a reaction mixture containing a 200 nM forward primer, 200 nM reverse primer, iTaq universal SyBR Green reaction mix, iScript reverse transcriptase, and 200 ng of total RNA. Real-time PCR was performed using the ABI PRISM 7700 Sequence Detection System , and data were analyzed using the ΔΔCT method with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the reference gene. The PCR primers used in this study are listed in TM assay and gene expression data were analyzed using two-way ANOVA tests followed by Tukey’s post hoc test. The significance level used in this study was 5%.All experiments were conducted in triplicate, and the results are presented as mean ± standard deviation. Statistical analysis and data processing were performed using Prism software (GraphPad Software 9). The alizarin red S assay results were subjected to one-way analysis of variance (ANOVA) testing, and Tukey’s multiple comparisons test was performed to compare the conditions. The AlamarBlueXPS measurements were performed on all samples, specifically at the C1s, N1s, and O1s core levels. The C1s spectra, as shown in The N1s spectra, as illustrated in The O1s spectra, as presented in The presence of characteristic N1s signals from the peptide indicates the immobilization of the peptide on the PEEK surface. The amount of peptide on the surface is comparable in both samples .As depicted in In Live and dead images confirm that HOB cells colonized all the test samples. Generally, on the tested surface, no dead cells (red cells) were detected after 48 h in culture, suggesting the absence of cytotoxic effects, while just a few red cells were observed on the PEEK-N3BMP sample with magnification 20× . The numCellular morphology and the adhesion on HOB cells were investigated on the functionalized scaffolds using SEM. Cells appeared to adhere to both the treated surfaces. On the plain PEEK sample a, cells Both functionalization conditions statically increased mineralization after 21 days in culture . A greatTM assay over a period of 1, 4, 7, 14, and 21 days post-seeding. A statistically significant increase in cell proliferation was observed for both the PEEK-AoaBMP and PEEK-N3BMP samples on the first day  and PEEK-N3BMP on day 1 a. PEEK-AGene expression analysis was performed to allow the detection and quantification of transcript proteins for specific genes of interest associated with bone formation. Gene regulation is an important part of cell and tissue development, with different genes being turned on and off during the different stages of development. It is well known that environmental and local factors can impact the genes expressed. Hence, in this study, the functionalized biomaterials PEEK-AoaBMP and PEEK-N3BMP were investigated for their potential to influence the enhancement of the expression of osteogenic genes.In this study, the covalent functionalization of PEEK’s surface with the bioactive peptide GBMP1α was realized to improve PEEK’s interactions with osteoblasts. The peptide was grafted onto the surface through two different chemical strategies: via oxime formation thanks to amino-oxy introduced in the bioactive sequence, or via the photoactivation of an azido group (present in the peptide sequence as well).The advantages of enriching PEEK via oxime with bioactive sequences (adhesive peptides mapped from Vitronectin) have already been highlighted in our previous work . The ancTM detected a significant more than 3-fold increase in the functionalized samples over the control on day 1. On days 4, 7, and 14, an increasing trend with functionalized PEEK compared to non-functionalized PEEK was observed, but this was not significant. By day 21, the proliferation rate was again significantly increased by functionalization.At 48 h, no cytotoxicity was induced by the peptide of the PEEK surface, as reported in live and dead assay results; a few dead cells were visualized on PEEK-N3BMP only. In fact, the functionalization of PEEK induced a better colonization by HOB cells, in particular on PEEK-AoaBMP, where cell adherence was observed to be homogeneous. Moreover, SEM showed that functionalized PEEK induced cells to adhere to the whole surface, forming layers, while the unfunctionalized PEEK did not. The visual information obtained through the live and dead and SEM assays was consistent with the proliferation tests. In fact, functionalization with GBMP1α with both amino-oxy and azido groups increased the rate of HOB proliferation. AlamarBlueTo understand the role of BMP2 protein in osteogenic differentiation, we validated the expression of candidate osteogenic gene markers: SOX9, Runx2, ALP and OCN. Sox9 is an SRY-related transcription factor required for the expression of cartilaginous genes in the developing skeletal system; hence, it is associated with bone formation. RUNX2 is known to regulate the proliferation of osteoblast progenitors and their differentiation into osteoblasts. ALP, an early marker of osteogenesis, is highly expressed in the cells of mineralized tissue and plays a critical role in the formation of hard tissue. ALP acts by increasing the local rate of inorganic phosphate and facilitates mineralization, as well as reducing the extracellular pyrophosphate concentration, an inhibitor of mineral formation. OCN, a later marker of differentiation, is expressed specifically by osteoblasts, and is the most abundant non-collagenous protein in bone, influencing matrix mineralization. In this study, PEEK-AoaBMP promoted a faster and greater expression of the genes SOX9 and ALP, since on day 1, it induced significant upregulation of these genes compared to the other conditions. It has been noted that the presence of SOX9 is detectable during the process of in vitro osteogenic differentiation of human mesenchymal stem cells, enhanced by the BMP-2 protein , while AThe regeneration of bone is a complex, well-orchestrated physiological process controlled by the local environment. In cases where the bone defect is large or compromised, biomaterials are often used to bridge the gap and provide a surface for the recruitment of cells. The biomaterial selection is dependent on the attitude to promote the migration, proliferation, and differentiation of bone cells, often functionalized to enhance this process. The use of PEEK has been well documented due to biocompatibility properties that avoid severe adverse reactions such as stress shielding; however, the great limitation of PEEK is its inability to integrate with the biological system. In this study, we have demonstrated that the functionalization of PEEK resulted in an enhancement of biological properties. Both chemical strategies, via oxime and via photoactivation, enhanced the activity of HOB cells on the surface of PEEK, with PEEK-N3BMP inducing a higher calcium deposition compared to PEEK-AoaBMP. It is well known that BMP-2 plays a significant role during embryonic development, and in bone remodeling and the maintenance of bone homeostasis in adulthood. The mechanism of action of BMP is via Smad; it acts as a signaling mediator in osteoblast–osteoclast coupling, which critically affects the rate of bone remodeling. Bone morphogenetic proteins can upregulate the expression of the inhibitory Smad proteins. These Smads are phosphorylated and translocate into the nucleus, where they regulate the transcription of target genes. A major function of BMP2 is the activation of specific osteogenic genes. However, to gain more insight and clarification on the mechanisms by which these peptides work, upcoming research will investigate their involvement in driving stem cells towards the osteogenic lineage and assess the regulatory signaling pathways in an in vitro setting.